Amplified isothermal detection of polynucleotides with atp release

Abstract

The presence of a target polynucleotide sequence of interest, including targets comprising genetic variations or a single nucleotide polymorphism, is detected by a DNA polymerization reaction, where the reaction mixture includes mixtures of nucleotides including at least one chimeric nucleoside tetraphosphate dimer ATP-linked nucleotide (ARN), in which ATP is the leaving group. DNA synthesis with ARNs is shown to be sequence specific, based on priming with a primer or template complementary to a target sequence. The released ATP is assayed in a qualitative or quantitative analysis, where one equivalent of ATP is released for every deoxynucleotide incorporated from an ARN.

What is claimed is: 
     
         1 . A method for detecting the presence of a target polynucleotide sequence in a sample comprising nucleic acids, the method comprising:
 contacting the nucleic acid with a reaction mixture comprising:   at least one ATP-releasing nucleotide (ARN) having a structure   
       
         
           
           
               
               
           
         
       
       wherein R is where R is any purine or pyrimidine, or an analog thereof that retains an ability to base pair with a complementary nucleotide; and optionally dNTPs, wherein the combination of dNTPs and ARN is sufficient to provide a substrate for all bases present in the sequence of interest;
 a primer or template complementary to a sequence of interest in the target polynucleotide; and 
 a DNA polymerase or reverse transcriptase that incorporates ARNs; and 
 detecting the presence of ATP released during extension of the primer or target by the DNA polymerase or reverse transcriptase. 
 
     
     
         2 . The method of  claim 1 , wherein R is selected from adenine, thymine, guanine, and cytosine. 
     
     
         3 . The method of  claim 1 , wherein the reaction mix comprises a single ARN and one or more dNTPs. 
     
     
         4 . The method of  claim 1 , wherein the reaction mixture comprises two or more different ARNs. 
     
     
         5 . The method of  claim 1 , wherein the reaction mixture comprises three of more different ARNs. 
     
     
         6 . The method of  claim 1 , wherein the reaction mixture comprises four different ARNs. 
     
     
         7 . The method of  claim 1 , wherein the reaction mix comprises one or both of dAppppA and dGppppA. 
     
     
         8 . The method of  claim 1 , wherein the reaction mix comprises one or both of dAppppA and dTppppA. 
     
     
         9 . The method of  claim 1 , wherein the reaction mixture comprises a DNA polymerase. 
     
     
         10 . The method of  claim 9 , wherein the reaction mixture comprises a reverse transcriptase. 
     
     
         11 . The method of  claim 9 , wherein the k cat  values for ARNs with the DNA polymerase or reverse transcriptase are within about 20-fold of those of native dNTPs. 
     
     
         12 . The method of  claim 1 , wherein the reaction mixture comprises a primer complementary to the sequence of interest, of from about 8 to about 35 nt. in length. 
     
     
         13 . The method of  claim 12 , wherein the complementary region of the primer is at least 90% identical to the sequence of interest. 
     
     
         14 . The method of  claim 1 , wherein the primer is complementary to an allelic form, where the terminal 3′ nucleotide of the primer is specific to a position of variation. 
     
     
         15 . The method of  claim 12 , wherein the primer comprises a region of non-complementarity to the sequence of interest. 
     
     
         16 . The method of  claim 1 , wherein the reaction mixture comprises a template comprising a region complementary to the sequence of interest. 
     
     
         17 . The method of  claim 16  wherein the complementary region of the primer is at least 90% identical to the sequence of interest. 
     
     
         18 . The method of  claim 16 , wherein the template is circular. 
     
     
         19 . The method of  claim 16 , wherein the target polynucleotide is less than about 25 nt. in length. 
     
     
         20 . The method of  claim 1 , wherein detecting ATP comprises the step of contacting the reaction mixture with luciferin and an ATP-dependent luciferase enzyme to produce light. 
     
     
         21 . The method of  claim 20 , wherein the luciferase is added to the reaction mix after a period of time sufficient to accumulate products of the polymerization reaction. 
     
     
         22 . The method of  claim 19 , wherein the luciferase is included in the initial reaction mixture. 
     
     
         23 . The method of  claim 1 , wherein detecting ATP comprises the step of contacting the reaction mixture with an ATP-responsive fluorescent dye. 
     
     
         24 . The method of  claim 14 , wherein the presence of ATP released during extension of the primer by the DNA polymerase is compared to the level of release from a primer for a different allele at the SNP, and wherein the release is at least 50% higher if the nucleic acids in the sample comprise the specific allele in the primer. 
     
     
         25 . A reaction mixture for use in a method of  claim 1 . 
     
     
         26 . A kit comprising at least one ARN and optional dNTP reagents for use in a method of  claim 1 . 
     
     
         27 . A method for synthesis of an ATP-releasing nucleotide (ARN) having a structure 
       
         
           
           
               
               
           
         
         wherein R is where R is any purine or pyrimidine, or an analog thereof, the method comprising:
 contacting salts of either (a) deoxynucleoside monophosphates (dNMPs) or (b) AMP with an activating agent; and 
 reacting the product of (a) with a salt of 5′-ATP or reacting the product of (b) with salts of a desired deoxynucleotide-5′-triphosphates (dNTP). 
 
       
     
     
         28 . The method of  claim 27 , wherein the activating agent is carbonyldiimidazole or a carbodiimide agent. 
     
     
         29 . The method of  claim 27 , wherein the salt is a tetra- or tri-alkylammonium salt.